The present invention relates, in general, to compositions, methods and diagnostic kits useful for the detection of fish-killing raphidophyte algae using rRNA-targeted probes.
Raphidophytes are algae of the class Raphidophyceae. Raphidophytes such as Heterosigma akashiwo (Hada) ex Sournia and Fibrocapsa japonica Toriumi and Takano are well known in temperate seas as causative agents for mass finfish kills in seapen aquaculture. For example, during the summer of 1989, a large bloom of Heterosigma akashiwo in Big Glory Bay, Stewart Island, New Zealand, caused extensive mortality of caged Quinnat salmon (Onchorynchus tshawytscha), the loss being valued at NZD $4.5 million [Chang F. H., Anderson, C. and Boustead, N. C. 1990. This event was the first record of a Heterosigma (Rapbidophyceae) bloom with associated mortality of cage-reared salmon in Big Glory Bay, New Zealand. NZ J. Mar. Freshwater. Res. 24: 461-469; MacKenzie L. 1991.
Harmful algal bloom research and monitoring has traditionally been based on ecological and microbiological measurements which are laborious, time-consuming, and reliant on experienced operators. The rapid identification and enumeration of harmful raphidophyte species is crucial for the management of cultured finfish, shellfish and wild resources in order to avoid stock loss.
Thus, there is a need to develop a test system for rapid, sensitive and cost effective analysis of Raphidophytes that permits as near as possible real time monitoring of the algae.
In order to meet these needs, the present invention is directed to compositions, methods and diagnostic kits useful for the detection of fish-killing raphidophyte algae using rRNA targeted probes. The probes comprise a segment of nucleic acid capable of selectively hybridizing, under selective hybridizing conditions, to large-subunit ribosomal RNA from raphidophytes.
The probes include those oligonucleotide probes having sequences selected from SEQ ID NO: 3 through SEQ ID NO: 23 and homologous sequences. The probes maybe utilized in various combinations including pairwise. The probes of the invention may be of the formula [X-Y-Z]n where X is a sequence of 0 to 100 nucleotides or nucleotide analogs that are non-homologous to conserved or nonconserved regions of raphidophyte nucleic acid. In the formula, Y is a sequence of 10 to 100 nucleotides or nucleotide analogs that are capable of hybridizing under hybridizing conditions to hypervariable regions of the ribosomal RNA of raphidophytes. Such sequences for Y include those sequences selected from SEQ ID NO:3 through SEQ ID NO:23 and homologous sequences. Furthermore, Z is a sequence of 0 to 100 nucleotides or nucleotide analogs that are non homologous to conserved or non conserved regions of raphidophyte nucleic acid. The sequence of Z may be the same or different from X. Finally, n is 1 to 500 or more.
In addition to compositions, methods are disclosed for the detection of raphidophytes from a marine sample using fluorescent in situ hybridization (F.I.S.H.) and sandwich hybridization assays (S.H.A.). These methods comprise the steps of: permeabilizing the species of raphidophyte to be assayed to expose the ribosomal RNA; contacting the exposed ribosomal RNA, under hybridizing conditions, with oligonynucleotide probes capable of selectively hybridizing to the hypervariable regions of the ribosomal RNA of at least one species of raphidophyte; and detecting hybridization complexes as an indication of the presence of the raphidophyte cell in the sample.
In addition to compositions and methods, there are disclosed herein diagnostic kits for use in determining the presence of raphidophytes which comprise a synthetic oligonucleotide probe complementary to the aforementioned hypervariable or conserved regions of the ribosomal RNA of a raphidophyte species from a marine sample. The kits may include hybridization buffers.
The present invention has utility in providing an easy, sensitive, and specific test for algae which may kill finfish and invertebrates.